Designer diatom episomes delivered by bacterial conjugationNature Communications

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Authors
Bogumil J. Karas, Rachel E. Diner, Stephane C. Lefebvre, Jeff McQuaid, Alex P.R. Phillips, Chari M. Noddings, John K. Brunson, Ruben E. Valas, Thomas J. Deerinck, Jelena Jablanovic, Jeroen T.F. Gillard, Karen Beeri, Mark H. Ellisman, John I. Glass, Clyde A. Hutchison III, Hamilton O. Smith, J. Craig Venter, Andrew E. Allen, Christopher L. Dupont, Philip D. Weyman
Year
2015
DOI
10.1038/ncomms7925
Subject
Chemistry (all) / Biochemistry, Genetics and Molecular Biology (all) / Physics and Astronomy (all)

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ARTICLE

Received 19 Dec 2014 | Accepted 13 Mar 2015 | Published 21 Apr 2015

Designer diatom episomes delivered by bacterial conjugation

Bogumil J. Karas1,w, Rachel E. Diner2,3, Stephane C. Lefebvre2, Jeff McQuaid2, Alex P.R. Phillips1,

Chari M. Noddings1, John K. Brunson1, Ruben E. Valas2, Thomas J. Deerinck4, Jelena Jablanovic2,

Jeroen T.F. Gillard2, Karen Beeri2, Mark H. Ellisman4, John I. Glass1, Clyde A. Hutchison III1, Hamilton O. Smith1,

J. Craig Venter1,2, Andrew E. Allen2,3, Christopher L. Dupont2 & Philip D. Weyman1

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and

Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

DOI: 10.1038/ncomms7925 OPEN 1 Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA. 2Microbial and Environmental Genomics Group, J. Craig

Venter Institute, La Jolla, California 92037, USA. 3 Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego,

La Jolla, California 92037, USA. 4National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, California 92093, USA. w Present address: Designer Microbes Inc., London, Ontario, Canada. Correspondence and requests for materials should be addressed to P.D.W. (email: pweyman@jcvi.org).

NATURE COMMUNICATIONS | 6:6925 | DOI: 10.1038/ncomms7925 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved.

D iatoms are eukaryotic phytoplankton that contribute a significant fraction of global primary productivity and demonstrate great potential for autotrophic bioproduction of fuels and higher value chemicals1–3. Although methods for genetic manipulation currently exist for some diatom species4–8, they are slow compared with the efficient methods available for other model microbes such as E. coli and yeast, and this has stymied both basic diatom research and applied strain development. To accelerate research in ecologically and biotechnologically important microalgae, we sought to develop diatom episomal vectors and improved transformation methods.

Circular DNA molecules have been previously isolated from diatoms, but they have never been successfully reintroduced as episomes9,10. This is unfortunate because episomes provide a reliable, consistent and predictable platform for protein expression by avoiding the complications of random chromosomal integration including multiple insertions, position-specific effects on expression and potential knockout of non-targeted genes4,7,8. Consistent protein expression from episomes may allow for efficient complementation experiments of diatom mutants made with recently developed TALEN technology11,12.

Episomes can be efficiently moved among bacteria and even between bacteria and eukaryotes via conjugation. Reports of transkingdom conjugation in other systems13–16 motivated us to explore direct conjugation of DNA into the pennate diatom

Phaeodactylum tricornutum as an alternative to biolistic transformation methods that are standard for many diatom species4–6. A conjugative link from Escherichia coli to diatoms streamlines the genetic manipulation workflow for diatoms.

Plasmids can be assembled and manipulated in vitro before being introduced in E. coli for direct conjugative transfer to diatoms, thus eliminating the need for time-consuming, high-yield plasmid

DNA preparations and access to expensive specific equipment and reagents (for example, gene gun). In conjugative systems, a two-plasmid system is often used where a conjugative plasmid (for example, RP4/RK2 or its derivatives such as pTA-MOB17) contains all of the genes required for establishing a conjugative bridge between the donor and recipient cell and a cargo plasmid contains the construct of interest. Inclusion of an origin of transfer (oriT) on the cargo plasmid allows it to be mobilized to the recipient by proteins encoded on the conjugative plasmid.

Maintenance of the cargo plasmid as an episome in the recipient cell further requires the presence of sequences that permit replication and segregation of the DNA molecule.

In this paper we describe our approach to identify a DNA sequence that can support episomal replication in the diatoms

Phaeodactylum tricornutum and Thalassiosira pseudonana.

Although we had initially intended to isolate a P. tricornutum sequence that supported episome replication in diatoms, we identified a sequence encoded by a yeast-derived sequence on the cloning vector that performed this function. We further developed a conjugation-based method to directly transfer episomes from E. coli to diatoms. These novel tools and methods compose an efficient and high-throughput system for diatom genetic manipulation that will enable rapid and fundamental advances in diatom functional genetics.

Results

Design of episomal vectors. To develop an extra-chromosomal replicating vector for diatoms, we first isolated a sequence that functions as a centromere or origin of replication in